Process of preparing vitamin k concentrates



v Patented Sept. 1942 vide aflvitaminjK-ccncentrate which will bring 7 K45 known tgbe present in I: tion of K concentrates. s

" -,.t i v mi ne s I Nonrawing.

The present invention relates to vitamin K. these-called anti-hemorrhagic or coagulation? vitamin.

od essential for normal 1 .Y-WYBOCEVSS or'rnnrlinnvavrmmlv K;

l Em ei 1 4 Byron Biegel and Perrin,G'.-' smith, Evanstonylll lf g your a. Schweitzer, Fromm-on, and Vernon c.

'Free, Northohicaganr, assiznors to Abbott laboratories, North. Chicago, Ill;,a corporation 1 Airman Ilecenilier.22, 193 9,

.SerialN oL31 0,542 7 onic- -31 vitamin K 4 concentrates; and more particularly to an im- I-"prOVed -method. for producing concentrates of .vitam n K out using commercially practical; recovery processes have shown the retentlonof-the" vitamin K I by the suggested adsorbatesto be suchthat at a vitamin K concentrate the efficiency of which I j is so greatthat a few milligrams to a few hundred (administered, for example, with or p without bile salts; orally o r-injected intramus-.

cularlmetcb Lwill' maintain the normal coagula tion time of blood. j

It is a' further object of this invention to ro thecoagulationftimelofblood back to normal and stopbleeding in patients suileringfrom disordersj conducive to low,prothrombin.content, such as -'hemorrhaglc diathesis of'jaundice or biliary tractv 5 well; as} in certain conditions or diseases where ther-iisinterference with intestinal absorption .of'vita'minK. 1

naturalsubstances, particularly in the green leaf portion of various plants-suchas alfalfa; young many cereals, 81 86111 thelike'. An extraction oi."

known solvents; for "examplegacetone, methyl alcohol, ethyl ether; etc. yields an extract contaii'iing the" desired. vitamin K, together with relatively large amounts; of 'unwanted'mate'rials;

' these vegetable materials with any of the well "including the; chlorophylls and. other pigments, v

' as, well as'other compounds such as the por- 'Dhyrins, carotenoidsfandphytosterols. Various processes have beensug'gested for separatingfthe inactivel(unwanted)-material s from'the vitamin, but most vof these have-beenfound to possess .vseriousdisadva'ntages and to be relatively unsatisfactoryior use -in the commercial producesium the plantmatter leave the desired our new process:

weight of the dry vegetable; a. I stirred until the supernatant liquid not smear; light yellow color. The mixture is I pymtiaticn.

A second extract is then formedby best only a portion of' the vitamin can be recovered from them.

After a prolonged rese ingf very weakly acidic) fuller'sfearthfirid powdered silicajg'el, possessed the unique and-um expected property of selectively adsorbingchloro-i I phylls and other pigmentsfrom-an extract containing' vitamin K withoutremov'ing (or atjy the most only insignificant amounts) jot-vitamin "K.: We also discovered thatithe .silicatescould I 1 I be used to remove large portions o'f'sterols; porphyrins and carotenoidsfrom decolqrlzed' 'extracts or from molecular distillatesobtain'ed therefrom I v without removing any-(oral; the most insigr'iiiician'i",i

amounts), of vitamin K. we'imf heridiscoveredthat-the silicates could be tofremoveyitamin activity from concentrates'including-pm'ifleds. concentrates and that thedesired vitamin'activ-f ity could be eluted and readilya recove'red'therefrom substantially free fromncontamination. by. J

sterols and other inert solids. Ej The following preferred erample w Example I The green leaf portion of variaus plants and the silicate completely removedspent"v silicate once or twice wi behind in extract. ,01" the. i"

* above 'mentionedadsorbents, however, have been a *found'to be unsatisfactory;-jfor-while-they serve f Ito. remove a large portion of inert/material in eluding pigments ,-from the, solvent "extract, they I x simultaneously removes-considerableportion of I I arch investigation we discovered that the silicates, particularly the' silicate compounds selected: from the, group consisting of magnesium tri-sili'cate, acidic (include .to contain vitamin K, preierably'alralfa'or-young" cereal plants, are first carefully dried anagmm'im v About fifteen hundred grams of dry-materialis then macerated or percolated withjabduf 'lit'ers' of a suitable solvent, preferablyjheiiane," the percolation being" continued most-jot", ,green pigments have been-extracted To this. diluteiextract is next added at'jtherate of approi'timatelyeigh pg centby mixture:

solvent such as hexane. The two extracts are then combined and distilled in vacuo' in the usual manner in order to remove the solvents.

'and'removal of the solvent, yields about 30 mm of an orange-red oil containing substantially all The quantity of magnesium trisillcate used in the above illustrative example may vary accord-' crease in potency.

The amountof fullers earth used in theabove of the vitamin K activity originally present in the vegetable material. This latter step is accompanied by a reduction in volume and an inexample may also vary according to the quantity of pigment in the vegetable material. The optimum amounts of fullers earth or dry silica gel,

- which gives similar results, may be determined be employed. In addition other'solvents such as methyl alcohol, ethyl alcohol, acetone, ethyl ether, benzol, etc. may be employed, although the use'of an aliphatic hydrocarbon solvent with the silicate is preferred as the removal 'of the green pigment is more readily effectedv in this type of solvent. If the other type of solvents e. g. alcohol, acetone, etc. are used initially, they may be removed in vacuo and the green residue subsequently taken up in a hydrocarbon solvent aside'at 0"'C. fora period of about 24 hours. 2

The precipitate formed by this treatment has no (preferably hexane) for.treatment with the adsorbent.

Dried alfalfa meal treated in accordance withthe above example yields a decolorized concentrate appearing as a dark reddish, yellow viscous oil containing substantially all of the vitamin K activity contained in the original vegetable material. This oil may be further purified by dissolving it in a fresh portion of organic solvent, preferably hexane, in the proportion of one liter of solvent for each ten grams of oil, and treating with additional silicate, e. g. magnesium trisilicate in amounts of approximately 5 per cent by weight of the original dry vegetable material. This treatment removes a large portion of the yellowish and reddish prophyrins and carotenoids as well as the sterols without removing the vitamin K activity. After removal of the organic solvent,.a concentrate is obtained which is 2-3 times the vitamin K potency of the original reddish, yellow oil.

The organic solvent extracts, whether crude or partly decolorized and purified, may be further concentrated by setting them aside, first at room temperature or in the refrigerator and later at zero degrees centigrade or below. This treatment precipitates additional portions of sterols which may be readily separated by filtration.

The following additional example will serve to illustrate the present invention.

Example II ance with the previously described example contains substantially all of the vitamin K contained in the original vegetable material.

The product obtained by the above treatment may be further purified by dissolving 40 grams of the oil concentrate in 500 c. c. of benzol and treating with about 180 grams of activated "Florex 1. e. fFlorex heated to 250 C. for approximately one hour and cooled. This additional treatment removes a large part of the reddish-yellow pigments andother impurities andupon filtration by preliminary laboratory experiment.

Regardless of which type of silicate adsorbent is employed, we have found that a further concentration may be eflected by dissolving the active, decolorized oil in acetone,'approximately one-fourth by volume of the weight of the dry vegetable material used, and setting the solution.

vitamin K activity and may be removed by filtration at 0 C. The acetone may be removed from-the filtrate in vacuo in the usual manner.

Our investigations show that the silicate adsorbents remove sterols from purified, decolorized concentrates or molecular distillates obtained therefrom more eiIectively than freezing alone. In view of this, in our preferred process we ordinarily. combine the two methods and remove sterols by subjecting the partially purified or decolorized concentrates to both freezing and additional silicate treatments. As an example,

" a decolorized concentrate (molecularly distilled at a pressure of 10- mm.) .is subjected to several freezing treatments in acetone solution and subjected to several fullers earth treatments in petroleum ether solution. About 20 to 22 per cent of solids based on the original weight of the distillate and consisting largely of phytosterols may be removed in this manner.

The silicate treatment may be carried .out at room temperature or the mixture may be heated to boiling under reflux, or the initial stirring with the silicate may be done at room temperature and the subsequent washings carried out by boiling under reflux. The silicate treatment may also be carried out by passing the extract through a column of the adsorbent according to the usual practice in chromatographic adsorption. Bioassays of various type extracts for vitamin K before and after removal of the pigments, etc. show that substantially no vitamin K activity is lost by the silicate treatment.

The silicate adsorbents may also be employed for removal by adsorption of vitamin K activity from concentrates. For example, vitamin K activity may be adsonbed from a solution of 5 grams of distillate (preferably virtually sterol-free and containing for example about 25,000 Peirce Dann units of vitamin K per gram) in 200 c. c. of petroleum ether with about 40 grams of fullers earth, i. e. activated fiorex. The adsorbed vitamin K in turn may be eluted from the adsorbent .by using five 250 c. c. portions of ether containing about 5 per cent methanol. The solvent free eluate contains one-half or less of the original inactive solids and is of increased vitamin K potency per mg. Several, fold enrichments may be obtained by this or like methods.

From the above it will be apparent (1) that the silicates may .be used to remove vegetable :pi-gments from vitamin K extracts without removing vitamin K activity; (2) that the silicates may be used to remove phytosterols, etc. from vitamin K extracts without removing vitamin K aaaaiaa activity, and (3) that the silicates may be used to adsorb vitamin K from vitamin K concentrates. From the above it will also be apparent (4) that vitamin K adsorbed by a silicate adsorbent may be eluted therefrom by treatment with an organic solvent. These properties which make the silicates satisfactory for use in the present invention, are essential for an improved commercial process in which vitamin K concen-- trates are obtained from natural sources.

In the specification and claims the term silicate is intended to include silica compounds such as silica gel as well as clay silicates such as fullers earth. The preferred adsorbents are magnesimn trisilicate, acidic fullers earth and pawdered silica gel.

It will be understood that the present invention is not limited to the above illustrative examples. All modifications coming within the scope and spirit of the present invention are intended to be covered by the following claims.

We claim:

1. The steps in the process of preparing improved vitamin K concentrates which comprises treating vegetable material having vitamin K activity with a stable organic solvent so as to form a dilute vitamin K extract containing vegetable pigments and sterols, adding to the extract thus obtained a silicate adsorbent, stirring the mixture thus formed until substantially all or the pigments have been removed from the extract, subiecting the extract to further silicate adsorbent treatment in order to remove substantial amounts of sterols, subjecting the resulting extract to further silicate treatment to adsorb the vitamin K activity, and then recovering the purified vitamin K activity therefrom by an elution treatment with an organic solvent and separating said solvent from said vitamin activity.

2. In a process of preparing vitamin K concentrates the steps which comprise treating vegetable material having vitamin K activity with a stable hydrocarbon solvent so as to form a dilute vitamin K extract containing vegetable pigments and sterols, adding to the extract thus obtained suflicient magnesium trisilicate to remove substantially all of the pigments from the extract, separating the extract from the adsorbent, and separating the solvent from said extract.

3. In a process of preparing vitamin K concentrates the steps which comprise treating vegetable material having vitamin K activity with a stable hydrocarbon solvent so as to form a dilute vitamin K extract containing vegetable pigments and sterols, adding to the extract thus obtainedsuflicient acidic fullers earth to remove substantially all of the pigments from the extract, separating the extract from the adsorbent, and separating the solvent from said extract.

4. In a process of preparing vitamin K concentrates the steps which comprise treating vegetable material having vitamin K activity with a stable hydrocarbon solvent so as to form a dilute vitamin K extract containing vegetable pigments and sterols, adding to the extract thus obtained suflicient powdered silica gel to remove substantially all of the pigments from the extract, separating the extract from the adsorbent, and separating the solvent from said extract.

5. The process of preparing vitamin K concentrates which comprises'treating ground dry alfalfa with hexane so as to form a dilute vitamin K extract containing pigments and sterols, adding to the extract thus obtained a small amount of magnesium trisilicate, stirring the mixture thus formed until substantially all of the pigments have been adsorbed by the trisilicate, separating the extract from the insoluble materiaL'and removing the hexane from the extract by distillation in v-acuo.

6. The process of claim 3, in which the extract is re-dissolved in acetone and cooled to about 0 C. in order to precipitate sterols.

'l. The process for obtaining an improved vitamin K concentrate which comprises treating a virtually sterol-free vitamin K concentrate with iullers earth so as to adsorb the vitamin K activity contained therein, and then recovering the purified vitamin K activity therefrom by an elution treatment with an organic solvent and separating said solvent from said vitamin activity.

BYRON RIEGEL. PERRIN G. SMITH. CARL E. SCHWEITZER. VERNON C. FREE. 

